Toxoplasma gondii is an obligate intracellular parasite that invaginates the host plasma membrane upon invasion to form a parasitophorous vacuole inside which the parasite replicates and survives. Like the related malaria parasite, Plasmodium falciparum, we recently discovered that T. gondii ingests host cytosolic proteins and targets them for degradation in a lysosome-like organelle called the vacuolar compartment or VAC. This discovery challenges the longstanding notion that T. gondii lacks endocytosis, because of its isolation in a non-fusogenic vacuole. Preliminary studies show that ingestion- deficient parasites display attenuated virulence and defective chronic infection in vivo. Further, these parasites are more vulnerable to killing by interferon gamma-dependent mechanisms, implying a role in immune evasion. Genetic and live cell imaging studies of intracellular parasites suggest a vesicular pathway for uptake from the intravacuolar network, a specialized structure in the parasitophorous vacuole. Immunofluorescence analysis indicates that ingested material traverses the late endosome of the parasite en route to the VAC. Our central hypothesis is that host protein-containing vesicles, generated from the intravacuolar network, are trafficked to the VAC via clathrin- mediated endocytosis. I have designed two specific aims to test this hypothesis. In Aim 1, I will use live-cell imaging to directly visualize vesicular trafficking fromthe parasitophorous vacuole membrane to the VAC to determine if the intravacuolar network is a conduit for ingestion. In Aim 2, I will use state of the art Stimulated Emission Depletion Microscopy to localize ingested host proteins within the parasite endosomal system of wild type parasites or parasites expressing dominant negative forms of suspected endocytic players to elucidate the pathway of trafficking within T. gondii.